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Invertebrates lack the specialized immune-memory cells responsible for vertebrate-like acquired immunity. However, there is increasing evidence that past infection by the same pathogen can ‘prime’ the insect immune response, resulting in improved survival upon reinfection. The mechanisms underlying these phenomenological accounts of priming are diverse, and often not completely clear. Here, we investigated the generality, specificity and mechanistic basis of immune priming in the fruit flyDrosophila melanogasterwhen infected with the gram-negative bacterial pathogenProvidencia rettgeri. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding. We find that priming inDrosophilais a long-lasting, pathogen-specific response, occurring in several fly genetic backgrounds and is particularly stronger in male flies. Mechanistically, we find that the enhanced survival of individuals primed with an initial non-lethal bacterial inoculum coincides with a transient decrease in bacterial loads, and that this is likely driven by the IMD-responsive antimicrobial-peptideDiptericin-Bin the fat body. Further, we show that whileDiptericinsare required as the effector of bacterial clearance, it is not solely sufficient for immune priming, and requires regulation by the peptidoglycan recognition proteinsPGRP-LB, PGRP-LCandPGRP-LE. We discuss potential explanations for the observed sex differences in priming, and discuss the epidemiological consequences of innate immune priming in invertebrates.Author summaryImmunisation using attenuated or inactivated pathogens remains one of the most successful public health practices to reduce the incidence of infectious diseases. Immunisation works because humans and other vertebrate animals have evolved an immune response capable of specific immune memory, which ensures a strong, precise, and effective response to a secondary infection. While invertebrates lack the vertebrate-like specialized immune memory cells responsible for acquired immunity, there is now substantial evidence that invertebrates are capable of immune priming, characterised by improved survival to a pathogenic infection following an initial exposure to an attenuated or inactivated pathogen. Here, we investigated the occurrence, generality, specificity and mechanistic basis of immune priming in fruit flies when infected with the gram-negative pathogenProvidencia rettgeri. We find that priming with an initial non-lethal bacterial inoculum results increased survival after a secondary lethal challenge with the same live bacterial pathogen, and that this protective response may last at least two weeks after the initial exposure, is particularly strong in male flies, and occurs in several genetic backgrounds. We also find that primed flies shed less pathogen during infection, suggesting that priming can be a useful tool to reduce pathogen transmission in insects.

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved among many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. Our results demonstrate that Drosophila strains from different locations differ in sleep and activity, and that females are generally more active than males. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we were able to quantify the among-line variance attributable to mitochondrial DNA, and we establish that mtDNA variation affects both activity and sleep, in a sex-specific manner. Altogether our study highlights the important role that mitochondrial genome variation plays on organismal physiology and behaviour.

Identifying how infection modifies host behaviours that determine social contact networks is important for understanding heterogeneity in infectious disease dynamics. Here, we investigate whether group social behaviour is modified during bacterial infection in fruit flies ( Drosophila melanogaster ) according to pathogen species, infectious dose, host genetic background and sex. In one experiment, we find that systemic infection with four different bacterial species results in a reduction in the mean pairwise distance within infected female flies, and that the extent of this change depends on pathogen species. However, susceptible flies did not show any evidence of avoidance in the presence of infected flies. In a separate experiment, we observed genetic- and sex-based variation in social aggregation within infected, same-sex groups, with infected female flies aggregating more closely than infected males. In general, our results confirm that bacterial infection induces changes in fruit fly behaviour across a range of pathogen species, but also highlight that these effects vary between fly genetic backgrounds and can be sex-specific. We discuss possible explanations for sex differences in social aggregation and their consequences for individual variation in pathogen transmission.

Dietary restriction (DR), limiting calories or specific nutrients without malnutrition, extends lifespan across diverse taxa. Traditionally, this lifespan extension has been explained as a result of diet-mediated changes in the trade-off between lifespan and reproduction, with survival favoured when resources are scarce. However, a recently proposed alternative suggests that the selective benefit of the response to DR is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, and DR individuals would be frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. We tested this by rearing outbred female fruit flies (Drosophila melanogaster) on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured with a sterile pinprick or unstressed. We monitored lifespan, fecundity and measures of ageing. DR extended lifespan and reduced reproduction irrespective of injury and infection. Infected flies on lower protein diets had particularly poor survival. Exposure to infection and injury did not substantially alter the relationship between diet and ageing patterns. These results do not provide support for lifespan extension under DR being a side effect of benign laboratory conditions. This article is protected by copyright. All rights reserved.

Genetic variation for both resistance and disease tolerance has been described in a range of species infected with bacterial, viral and fungal pathogens. In Drosophila melanogaster, genetic variation in mortality following systemic Drosophila C Virus (DCV) infection has been shown to be driven by large effect polymorphisms in the viral restriction factor pastrel (pst). However, it is unclear if pst impacts variation in DCV titres (i.e. resistance), or if it also contributes to disease tolerance. We investigated systemic infection across a range of DCV challenge doses spanning nine orders of magnitude, in males and females of ten Drosophila Genetic Reference Panel (DGRP) lines carrying either a susceptible (S) or resistant (R) pst allele. Our results uncover among-line variation in fly survival, viral titers, and disease tolerance measured both as the ability to maintain survival (mortality tolerance) and reproduction (fecundity tolerance). We confirm the role of pst in resistance, as fly lines with the resistant (R) pst allele experienced lower viral titers, and we uncover novel effects of pst on host vigor, as flies carrying the R allele exhibited higher survival and fecundity even in the absence of infection. Finally, we found significant variation in the expression of the JAK-STAT ligand upd3 and the epigenetic regulator of JAK-STAT G9a. While G9a has been previously shown to mediate tolerance of DCV infection, we found no correlation between the expression of either upd3 or G9a on fly tolerance or resistance. Our work highlights the importance of both resistance and tolerance in viral defence.

Identifying how infection modifies host behaviours that determine social contact networks is important for understanding heterogeneity in infectious disease dynamics. Here, we investigate whether group social behaviour is modified during bacterial infection in Drosophila melanogaster, an established system for behavioural genetics, according to pathogen species, infectious dose, host genetic background and sex. We find that systemic infection with four different bacterial species results in a reduction in the mean pairwise distance within infected flies, and that the extent of this change depends on the infectious dose in a pathogen-specific way. In the presence of infected conspecifics, susceptible flies also tended to aggregate throughout time, but did not show any evidence of avoiding infected flies. We also observed genetic- and sex-based variation in social aggregation, with infected female flies aggregating more closely than infected males. In general, our results confirm that bacterial infection induces changes in fruit fly behaviour across a range of pathogen species, but also highlight that these effects vary between fly genetic backgrounds and can be sex-specific. We discuss possible explanations for sex differences in social aggregation and their consequences for individual variation in pathogen transmission.

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved between many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. The results demonstrate that Drosophila strains from different locations differ in sleep and activity, and the extent of variation differs between sexes, females in general being more active. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we establish that mtDNA variation affects both traits, sex specifically. Furthermore, by using previously published mtDNA copy number data, we detected a positive correlation between mtDNA copy number and the activity levels of the cybrid flies. Altogether, our study shows that both mtDNA variation and mitonuclear interactions affect activity and sleep patterns, highlighting the important role that both genomes play on life-history trait evolution.

The insect gut is frequently exposed to pathogenic threats and must not only clear these potential infections, but also tolerate relatively high microbe loads. In contrast to the mechanisms that eliminate pathogens, we currently know less about the mechanisms of disease tolerance. We investigated how well-described mechanisms that prevent, signal, control or repair damage during infection contribute to the phenotype of disease tolerance. We established enteric infections with the bacterial pathogen Pseudomonas entomophila in transgenic lines of Drosophila melanogaster fruit flies affecting dcy (a major component of the peritrophic matrix), upd3 (a cytokine-like molecule), irc (a negative regulator of reactive oxygen species) and egfr 1 (epithelial growth factor receptor). Flies lacking dcy experienced the highest mortality, while loss of function of either irc or upd3 reduced tolerance in both sexes. The disruption of egfr 1 resulted in a severe loss in tolerance in male flies but had no substantial effect on the ability of female flies to tolerate P. entomophila infection, despite carrying greater microbe loads than males. Together, our findings provide evidence for the role of damage limitation mechanisms in disease tolerance and highlight how sexual dimorphism in these mechanisms could generate sex differences in infection outcomes.

Disease tolerance describes a hosts ability to maintain health independently of the ability to clear microbe loads. However, we currently know little about the mechanisms that underlie disease tolerance or how known mechanisms of tissue damage signalling and repair may contribute to variation in tolerance. The Jak/Stat pathway plays a pivotal role in Drosophila humoral innate immunity, signalling tissue damage and triggering cellular renewal, making it a potential mechanism underlying the disease tolerance phenotype. Here, we show that disrupting the Jak/Stat pathway in Drosophila melanogaster alters disease tolerance during Pseudomonas entomophila systemic infection. Overall, flies with disrupted Jak/Stat show variation in survival that is not explained by variation in pathogen loads. For instance, mutations disrupting the function of ROS producing dual oxidase (duox) or the negative regulator of Jak/Stat, Socs36E render males less tolerant to systemic bacterial infection but not females. We also investigated whether the negative regulator of Jak/Stat, G9a which has previously been associated with tolerance of viral infections is also implicated in tolerance of bacterial infection. While female flies lacking G9a showed higher mortality and reduced bacterial clearance, disease tolerance did not differ between G9a mutants and the wildtype. This suggests that G9a does not affect tolerance during systemic bacterial infection as it appears to do with viral infection. Overall, our findings highlight that Jak/Stat signalling mediates disease tolerance during systemic bacterial infection and that this response differs between males and females. Our work therefore suggests that differences in Jak/Stat mediated disease tolerance may be a potential source of sexually dimorphic response to infection in Drosophila.

Disease tolerance is an infection phenotype where hosts show relatively high health despite harbouring elevated pathogen loads. Compared to the mechanisms of immune clearance our knowledge of the mechanisms underlying increased tolerance remains incomplete. Variation in the ability to reduce immunopathology may explain why some hosts can tolerate higher pathogen burdens with reduced pathology. Negative immune regulation would therefore appear to be a clear candidate for a mechanism underlying disease tolerance but this has not been tested directly for bacterial infections. Here, we examined how the negative regulation of the immune deficiency (IMD) pathway affects disease tolerance in Drosophila melanogaster when infected with the gram-negative bacterial pathogen Pseudomonas entomophila. We find that UASRNAi-mediated reduced expression of the negative regulators of IMD (pirk and caudal) severely reduced the ability to tolerate infection in both males and females across a wide range of infectious doses. While flies unable to regulate the IMD response exhibited higher expression of antimicrobial peptides and lower bacterial loads as expected, this was not accompanied by a proportional reduction in mortality. Instead, tolerance (measured as fly survival relative to its microbe load) was drastically reduced, likely due to the combination of increased immunopathology and cytotoxicity of elevated AMP expression. Our results therefore highlight that in addition to regulating an efficient pathogen clearance response, negative regulators of IMD also contribute to disease tolerance.

Parental care is a key component of an organism’s reproductive strategy that is thought to trade-off with allocation toward immunity. Yet, it is unclear how caring parents respond to pathogens: do infected parents reduce care as a sickness behavior or simply from being ill or do they prioritize their offspring by maintaining high levels of care? To address this issue, we investigated the consequences of infection by the pathogen Serratia marcescens on mortality, time spent providing care, reproductive output, and expression of immune genes of female parents in the burying beetle Nicrophorus vespilloides. We compared untreated control females with infected females that were inoculated with live bacteria, immune-challenged females that were inoculated with heat-killed bacteria, and injured females that were injected with buffer. We found that infected and immune-challenged females changed their immune gene expression and that infected females suffered increased mortality. Nevertheless, infected and immune-challenged females maintained their normal level of care and reproductive output. There was thus no evidence that infection led to either a decrease or an increase in parental care or reproductive output. Our results show that parental care, which is generally highly flexible, can remain remarkably robust and consistent despite the elevated mortality caused by infection by pathogens. Overall, these findings suggest that infected females maintain a high level of parental care, a strategy that may ensure that offspring receive the necessary amount of care but that might be detrimental to the parents’ own survival or that may even facilitate disease transmission to offspring.

Parental care is a key component of an organism’s reproductive strategy that is thought to trade-off with allocation towards immunity. Yet it is unclear how caring parents respond to pathogens: do infected parents reduce their amount of care as a sickness behaviour or simply from being ill, or do they prioritise their offspring by maintaining high levels of care? Here we explored the consequences of infection by the pathogenSerratia marcescenson mortality, time spent providing care, reproductive output, and expression of immune genes of female parents in the burying beetleNicrophorus vespilloides. We compared untreated control females with infected females that were inoculated with live bacteria, immune-challenged females that were inoculated with heat-killed bacteria, and injured females that were injected with buffer. We found that infected and immune-challenged females mounted an immune response and that infected females suffered increased mortality. Nevertheless, infected and immune-challenged females maintained their normal level of care and reproductive output. There was thus no evidence that infection led to either a decrease or an increase in parental care or reproductive output. Our results show that parental care, which is generally highly flexible, can remain remarkably robust and consistent despite the elevated mortality caused by infection by pathogens. Overall, these findings suggest that infected females maintain a high level of parental care; a strategy that may ensure that offspring receive the necessary amount of care but that might be detrimental to the parents’ own survival or that may even facilitate disease transmission to offspring.

Dietary restriction (DR), limiting calories or specific nutrients, extends lifespan across diverse taxa. This lifespan extension has been explained as diet-mediated changes in the trade-off between lifespan and reproduction, with survival favoured with scarce resources. Another evolutionary hypothesis suggests the selective benefit of the response is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, where DR individuals are frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. We tested this by rearing outbred female Drosophila melanogaster on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured or unstressed. We monitored lifespan, fecundity and ageing measures. DR extended lifespan and reduced reproduction irrespective of injury and infection. These results do not support lifespan extension under DR being a side effect of benign laboratory conditions.

Infomration about the creation and maintenance of Ashworth outcrossed DGRP population

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

To stay healthy, animals should avoid sources of infection. However, some important behaviors, such as foraging and reproduction, can increase infection risk. We tested experimentally whether fruit flies avoided infected food sources when foraging as larva and deciding where to lay eggs as adults. Larvae did not avoid infected food when foraging. Mothers however, sometimes avoided laying eggs on infectious food., Deciding where to eat and raise offspring carries important fitness consequences for all animals, especially if foraging, feeding, and reproduction increase pathogen exposure. In insects with complete metamorphosis, foraging mainly occurs during the larval stage, while oviposition decisions are made by adult females. Selection for infection avoidance behaviors may therefore be developmentally uncoupled. Using a combination of experimental infections and behavioral choice assays, we tested if Drosophila melanogaster fruit flies avoid infectious environments at distinct developmental stages. When given conspecific fly carcasses as a food source, larvae did not discriminate between carcasses that were clean or infected with the pathogenic Drosophila C Virus (DCV), even though cannibalism was a viable route of DCV transmission. When laying eggs, DCV-infected females did not discriminate between infectious and noninfectious carcasses, and laying eggs near potentially infectious carcasses was always preferred to sites containing only fly food. Healthy mothers, however, laid more eggs near a clean rather than an infectious carcass. Avoidance during oviposition changed over time: after an initial oviposition period, healthy mothers stopped avoiding infectious carcasses. We interpret this result as a possible trade-off between managing infection risk and maximizing reproduction. Our findings suggest infection avoidance contributes to how mothers provision their offspring and underline the need to consider infection avoidance behaviors at multiple life-stages.

Deciding where to eat and raise offspring carries important fitness consequences for all animals, especially if foraging, feeding and reproduction increase the risk of exposure to pathogens. In insects with complete metamorphosis, foraging occurs mainly during the larval stage, while oviposition decisions are taken by adult-stage females. Selection for infection avoidance behaviours may therefore be developmentally uncoupled. Using a combination of experimental infections and behavioural choice assays, here we tested if Drosophila melanogaster fruit flies avoid potentially infectious environments at distinct developmental stages. When given conspecific fly carcasses as a food source, larval-stage flies did not discriminate between carcasses that were clean or infected with the pathogenic Drosophila C Virus (DCV), even though scavenging was a viable route of DCV transmission. Adult females however, discriminated between different oviposition sites, laying more eggs near a clean rather than an infectious carcass if they were healthy; DCV-infected females did not discriminate between the two environments. While potentially risky, laying eggs near potentially infectious carcasses was always preferred to sites containing only fly medium. Our findings suggest that infection avoidance can play an important role in how mothers provision their offspring, and underline the need to consider infection avoidance behaviours at multiple life-stages.

Viruses are major evolutionary drivers of insect immune systems. Much of our knowledge of insect immune responses derives from experimental infections using the fruit fly Drosophila melanogaster. Most experiments, however, employ lethal pathogen doses through septic injury, frequently overwhelming host physiology. While this approach has revealed several immune mechanisms, it is less informative about the fitness costs hosts may experience during infection in the wild. Using both systemic and oral infection routes we find that even apparently benign, sub-lethal infections with the horizontally transmitted Drosophila C Virus (DCV) can cause significant physiological and behavioural morbidity that is relevant for host fitness. We describe DCV-induced effects on fly reproductive output, digestive health, and locomotor activity, and we find that viral morbidity varies according to the concentration of pathogen inoculum, host genetic background and sex. Notably, sub-lethal DCV infection resulted in a significant increase in fly reproduction, but this effect depended on host genotype. We discuss the relevance of sub-lethal morbidity for Drosophila ecology and evolution, and more broadly, we remark on the implications of deleterious and beneficial infections for the evolution of insect immunity.

Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. Wolbachia-mediated protection against viral infection has been extensively demonstrated in Drosophila. In mosquitoes that are artificially transinfected with Drosophila melanogaster Wolbachia (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila to date. Here, we show that the route of infection is key for Wolbachia-mediated antibacterial protection. Drosophila melanogaster carrying Wolbachia showed reduced mortality during enteric—but not systemic—infection with the opportunist pathogen Pseudomonas aeruginosa. Wolbachia-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide Attacin A, and also increased expression of a reactive oxygen species detoxification gene (Gst D8). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection.

Viruses are major evolutionary drivers of insect immune systems. Much of our knowledge of insect immune responses derives from experimental infections using the fruit flyDrosophila melanogaster.Most experiments, however, employ lethal pathogen doses through septic injury, frequently overwhelming host physiology. While this approach has revealed a number of immune mechanisms, it is less informative about the fitness costs hosts may experience during infection in the wild. Using both systemic and oral infection routes we find that even apparently benign, sub-lethal infections with the horizontally transmitted Drosophila C Virus (DCV) can cause significant physiological and behavioral morbidity that is relevant for host fitness. We describe DCV-induced effects on fly reproductive output, digestive health, and locomotor activity, and we find that viral morbidity varies according to the concentration of pathogen inoculum, host genetic background and sex. Notably, sub-lethal DCV infection resulted in a significant increase in fly reproduction, but this effect depended on host genotype. We discuss the relevance of sub-lethal morbidity forDrosophilaecology and evolution, and more broadly, we remark on the implications of deleterious and beneficial infections for the evolution of insect immunity.

Wolbachia-mediatedprotection against viral infection has been extensively demonstrated in Drosophila and in mosquitoes that are artificially inoculatedwith D. melanogaster Wolbachia (wMel), but to date no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila.Here we show that D. melanogaster carrying wMel shows reduced mortality during enteric – but not systemic - infection with the opportunist pathogen Pseudomonas aeruginosa, and that protection is more pronounced in male flies. Wolbachia-mediated protection is associated with increased early expression of the antimicrobial peptide attacinA, followed by increased expression of a ROS detoxification gene (gstD8), and other tissue damage repair genes which together contribute to greater host resistance and disease tolerance. These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic.

Parents can increase the fitness of their offspring by allocating nutrients to eggs and/or providing care for eggs and offspring. Although we have a good understanding of the adaptive significance of both egg size and parental care, remarkably little is known about the co-evolution of these two mechanisms for increasing offspring fitness. Here, we report a parental removal experiment on the burying beetle Nicrophorus vespilloides in which we test whether post-hatching parental care masks the effect of egg size on offspring fitness. As predicted, we found that the parent's presence or absence had a strong main effect on larval body mass, whereas there was no detectable effect of egg size. Furthermore, egg size had a strong and positive effect on offspring body mass in the parent's absence, whereas it had no effect on offspring body mass in the parent's presence. These results support the suggestion that the stronger effect of post-hatching parental care on offspring growth masks the weaker effect of egg size. We found no correlation between the number and size of eggs. However, there was a negative correlation between larval body mass and brood size in the parent's presence, but not in its absence. These findings suggest that the trade-off between number and size of offspring is shifted from the egg stage towards the end of the parental care period and that post-hatching parental care somehow moderates this trade-off.